Characterization of proteins often requires the ability to incorporate detectable groups—e.g., fluorochromes, chromophores, spin labels, radioisotopes, paramagnetic atoms, heavy atoms, haptens, crosslinking agents, and cleavage agents—at specific, defined sites. For proteins that do not contain pre-existing cysteine residues, site-specific labeling can be accomplished by use of site-directed mutagenesis to introduce a cysteine residue at the site of interest, followed by cysteine-specific chemical modification to incorporate the labeled probe. However, for proteins that contain pre-existing cysteine residues, site-specific labeling is difficult. Multiple strategies have been reported: (i) intein-mediated labeling (“expressed protein ligation”), (Muir, et al., Proc. Nat'l. Acad. Sci. USA, 95:6705-6710 (1998)); (ii) transglutaminase-mediated labeling (Sato et al., Biochem. 35:13072-13080 (1996)); (iii) oxidation-mediated labeling (Geoghegan, et al., Bioconj. Chem., 3:138-146 (1992)); and (iv) trivalent-arsenic-mediated labeling (Griffin et al., Science 281:269-272, 1998) (U.S. Pat. No. 6,008,378). Strategies (i)-(iii) do not permit in situ labeling (i.e., direct labeling of proteins in cuvettes, gels, blots, or biological samples—without the need for a subsequent purification step) or in vivo labeling (i.e., direct labeling of proteins in cells). Strategy (iv) requires a structural scaffold presenting two trivalent-arsenic atoms in a precisely defined spatial relationship and therefore relates only to a limited number of detectable groups (such as those having a detectable xanthene, xanthanone, or phenoxazinestructural nucleus).
Transition-metal chelates consisting of a transition-metal ion, such as Ni2+, Co2+, Cu2+, or Zn2+, in complex with a tridentate or tetradentate chelating ligand, such as iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA), exhibit high affinity for oligohistidine sequences, particularly hexahistidine sequences (Sulkowski, E., Trends Biotechnol., 3:1-7 (1985); Hochuli, et al., J. Chromat. 411:177-184 (1987); Hochuli, E. et al. BioTechnol. 6:1321-1325 (1988). FIG. 1 shows a proposed model for binding of a neighboring hexahistidine residue to a Ni-NTA resin as disclosed in Crowe, J. et al., Methods Mol. Biol., 31:371-387 (1994)).
The high affinity of interactions between transition-metal chelates and oligohistidine sequences, particularly hexahistidine sequences, has been verified using force microscopy experiments, which permit direct measurement of interaction forces on the single-molecule level and direct observation of molecular recognition of a single receptor-ligand pair (Kienberger, F. et al. Single Mol. 1:59-65 (2000); Schmitt, L. et al. Biophys. J. 78: 3275-3285 (2000)).
The high affinity of interactions between transition-metal chelates and oligohistidine sequences, particularly hexahistidine sequences, has been used advantageously to purify biomolecules containing, or modified to contain, “oligohistidine tags,” particularly “hexahistidine tags” (Hochuli, E. et al. BioTechnol. 6:1321-1325 (1988); Crowe, J. et al., Methods Mol. Biol., 31:371-387 (1994)). In this application, termed “immobilized-metal-chelate affinity chromatography,” a transition-metal chelate consisting of a transition-metal ion, such as Ni2+, Co2+, Cu2+, or Zn2+, in complex with a tridentate or tetradentate chelating ligand, such as iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA), is immobilized on a solid phase, such as chromatographic resin, and the resulting immobilized metal chelate is used to bind, and thereby purify from other components, tagged biomolecules.
The high affinity of interactions between transition-metal chelates and oligohistidine tags, particularly hexahistidine tags, also has been used advantageously in biosensor analysis of biomolecules containing, or modified to contain, oligohistidine tags, particularly hexahistidine tags (Gershon, et al. J. Immunol. Meths. 183:65-76 (1995); Nieba, L. et al. Anal. Biochem. 252:217-228 (1997)). Kienberger et al., Single Mol. 1; S9-65 (2000). In this application, a transition-metal chelate consisting of a transition-metal ion, such as Ni2+, Co2+, Cu2+, or Zn2+, in complex with a tridentate or tetradentate chelating ligand, such as iminodiacetic acid (IDA) or nitrilotriacetic acid (NTA), is immobilized on a biosensor chip, such a surface-plasmon-resonance biosensor chip, and the resulting immobilized metal chelate is used to detect, quantify, and analyze tagged biomolecules.
It would be advantageous to be able to use the high affinity of interactions between transition-metal chelates and oligohistidine tags, particularly hexahistidine tags, in labeling and in situ detection of tagged target materials, in particular, biomolecules.
There is a need for improved methods and compositions for protein labeling. In particular, there is a need for methods and compositions that permit in situ labeling, that permit in vivo labeling, and that encompass a wide range of detectable groups with different properties.